Multimodal Imaging of Fibrin

ABSTRACT

Fibrin-specific imaging agents that contain at least two imaging reporters are described, as well as methods of making and using the contrast agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/256,795, filed May 3, 2012, which is a 371 application ofInternational Application No. PCT/US2010/031396, filed on Apr. 16, 2010,and claims priority to U.S. Application Ser. No. 61/170,345, filed onApr. 17, 2009, which are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

This disclosure relates to fibrin-specific imaging agents having two ormore imaging reporters.

BACKGROUND

Fibrin is a major component of blood clots. Fibrin is a fibrillarprotein derived from the soluble plasma protein fibrinogen. During theclotting cascade the enzyme thrombin cleaves fibrinopeptides onfibrinogen resulting in an end-to-end polymerization to give the polymerfibrin. The transglutaminase Factor XIII further stabilizes the fibrinmesh by crosslinking the fibrin fibrils. Fibrin is found in all thrombi:fresh thrombus, old thrombus; thrombus in the venous system, arterialsystem, and cardiac chambers. In this respect fibrin is different fromactivated platelets which are found mainly in fresh thrombi in thearterial system. Besides thrombus, fibrin is often associated with solidtumors, and is found in the tumor stroma. Fibrin is often associatedwith complex atherosclerotic plaque where it is believed to be presentas a result of plaque rupture or fissure and subsequent healing.

Fibrin is a broadly useful target for imaging. For instance, sincefibrin is found in thrombus but not in the circulating blood, afibrin-targeted imaging would be expected to have high specificity fordisease. The concentration of circulating fibrinogen is on the order of1.5-4.0 g/L (4-12 μM), and one expects similar and higher levels offibrin in thrombus. High concentrations of fibrin, coupled with itspresence in all types of thrombi suggest that a fibrin-targeted imagingwould have high sensitivity for disease as well.

Because of the perceived sensitivity and specificity for disease, andthe relevance of fibrin in thromboembolic disease, cancer, andvulnerable atherosclerotic plaque, there have been several efforts todevelop fibrin-specific imaging probes. This large body of scientificand patent literature speaks to the still unmet medical need of imagingfibrin in order to detect, diagnose, or monitor therapy forthromboembolic diseases and cancer. Despite the innovative approaches toimaging fibrin, there remains room for improvement.

SUMMARY

This disclosure provides peptide-targeted imaging probes that contain atleast two imaging reporters. An imaging reporter (IR) is a group thatmakes the probe visible in a particular imaging modality. For instance,an IR can be a complex of Gd(III) to make the probe detectable by MRI,it can be a radionuclide to make the probe detectable by PET or SPECTimaging, or it can be a fluorophore detectable by optical imaging. Thetwo or more imaging reporters can be the same or can be different. Ininstances where the imaging reporters differ, it can be possible todetect the probe using two different imaging modalities. Surprisingly,appending different imaging reporters to a short fibrin specific peptidedid not affect the affinity of the resultant probe to fibrin.

An imaging agent, or a pharmaceutically acceptable salt form thereof,can include a fibrin binding peptide bound optionally through one ormore linkers to two or more imaging reporters, wherein at least one ofthe imaging reporters comprises the radioisotope of copper-64 (⁶⁴Cu,Cu-64).

A fibrin binding peptide includes any peptide known in the art to havean affinity for fibrin. In some embodiments, the fibrin binding peptidecomprises:

wherein:X₁ is selected from the group consisting of:

X₂ is selected from the group consisting of:

X₃ is selected from the group consisting of H and OH;X₄ is selected from the group consisting of H, I, Br, and Cl;X₅ is selected from the group consisting of H and CH₂COOH;X₆ is selected from the group consisting of:

andX₇ is selected from the group consisting of CH₂CH₂C(O)NH₂ andCH₂CH(CH₃)₂.

Any suitable linker can be used to link an imaging reporter to a fibrinbinding peptide. Examples include:

-   —NHCH(R)C(O)—, wherein R is any natural amino acid side chain;-   —NH(CH₂)_(n)C(O)—, wherein n is an integer from 1-6;-   —NHCH₂CH₂OCH₂CH₂C(O)—;-   —NHCH₂CH₂OCH₂CH₂OCH₂CH₂C(O)—;-   —NHCH₂C₆H₄CH₂NH—;-   —NH(CH₂)_(m)NH—, wherein m is an integer from 2-6;-   —NHCH₂OCH₂NH—;-   —NHCH₂CH₂OCH₂CH₂NH—; and-   —NHCH₂CH₂OCH₂CH₂OCH₂CH₂NH—.

The imaging reporters can be chosen from a chelator comprising aradioactive metal ion, a chelator comprising a paramagnetic metal ion,and a fluorescent dye. The two or more imaging agents can be the same orcan be different. In some embodiments, when the imaging reporterincludes a chelator, the chelator can be selected from the groupconsisting of:

wherein M is a radioactive metal ion or a paramagnetic metal ion.Radioactive metal ions can be, for example, ⁴⁵Ti, ⁵¹Mn, ⁵²Mn, ^(52m)Mn⁵²Fe, ⁶⁰Cu, ⁶¹Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc,^(99m)Tc, ¹¹⁰In, ¹¹¹In, ¹¹³In, ¹⁷⁷Lu. In some embodiments, theradioactive metal ion can be selected from the group consisting of:⁵²Fe, ⁶⁰Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ^(94m)Tc, ^(99m)Tc, ¹¹⁰In, and¹¹¹In. Paramagnetic metal ions can be, for example, a paramagnetic metalion having atomic numbers 21-29, 43, 44, and 57-83, such as Gd³⁺, Mn²⁺,Fe³⁺, and Mn³⁺.

An imaging agent can also be a fluorescent dye, such as

wherein:n is an integer from 0 to 4;each m is independently an integer from 0 to 6; andX is selected from the group consisting of SO₃ ⁻, OH, PO₄ ²⁻.

An example of a fibrin-specific imaging agent includes:

or a pharmaceutically acceptable salt form thereof.

Also provided herein is an imaging agent having the formula

IR₁-L₁-FTP-L₂-IR₂

or a pharmaceutically acceptable salt form thereof,wherein:FTP is a fibrin binding peptide;L₁ and L₂ are optional linkers;IR₁ and IR₂ are imaging reporters wherein the two imaging reporters areselected from the pairs consisting of: a fluorescent dye and a chelatorcomprising a radioactive metal ion; a fluorescent dye and a chelatorcomprising a paramagnetic metal ion; and a chelator comprising aradioactive metal ion and a chelator comprising a paramagnetic metalion.

For example, the imaging agent can be a compound of the formula:

or a pharmaceutically acceptable salt form thereof,wherein:X₁ is selected from the group consisting of:

X₂ is selected from the group consisting of:

X₃ is selected from the group consisting of H and OH;X₄ is selected from the group consisting of H, I, Br, and Cl;X₅ is selected from the group consisting of H and CH₂COOH;X₆ is selected from the group consisting of:

andX₇ is selected from the group consisting of CH₂CH₂C(O)NH₂ andCH₂CH(CH₃)₂.

In some embodiments, L₁ can be absent or can be selected from the groupconsisting of:

—NHCH(R)C(O)—, wherein R is any natural amino acid side chain;—NH(CH₂)_(n)C(O)—, wherein n is an integer from 1-6;—NHCH₂CH₂OCH₂CH₂C(O)—; and—NHCH₂CH₂OCH₂CH₂OCH₂CH₂C(O)—.In some embodiments, L₂ can be selected from the group consisting of:—NHCH₂C₆H₄CH₂NH—;—NH(CH₂)_(m)NH—, wherein m is an integer from 2-6;—NHCH₂OCH₂NH—;—NHCH₂CH₂OCH₂CH₂NH—; and—NHCH₂CH₂OCH₂CH₂OCH₂CH₂NH—.

Examples of imaging agents having two different imaging reportersinclude:

wherein M is a radioactive or paramagnetic metal ion.

Further provided herein is an imaging agent having the formula:

or a pharmaceutically acceptable salt form thereof,wherein:each M is independently a paramagnetic metal ion or Gd³⁺, with theproviso that no more than three M are Gd³⁺. In some embodiments, atleast one M is selected the group consisting of: ⁶⁴Cu, ⁶⁸Ga, ^(99m)Tc,and ¹¹¹In; for example, at least one M is ⁶⁴Cu.

The imaging agents described above can be combined with apharmaceutically acceptable carrier.

Methods of imaging fibrin in a mammal are also provided. In some cases,the method includes: a) administering to the mammal an imaging agenthaving at least one chelator comprising a paramagnetic metal ion; b)acquiring an image of the fibrin of the mammal using a nuclear imagingtechnique (e.g., single photon emission computed tomography and positronemission tomography); c) acquiring an anatomical image of the mammalusing magnetic resonance imaging or computed tomography; and d)overlaying the images of steps b) and c) to localize the image of fibrinwithin the anatomical image of the mammal.

In some embodiments, the images of steps b) and c) are acquiredsimultaneously. The method can further include administering to themammal a second imaging agent, wherein the second imaging agent does nottarget fibrin. For example, the second imaging agent is selected fromthe group consisting of: gadoteridol, gadopentetate, gadobenate,gadoxetic acid, gadodiamide, gadoversetamide, gadoversetamide, andgadofosveset for use with MRI and iopamidol, iohexol, ioxilan,iopromide, iodixanol, ioxaglate, metrizoate, and diatrizoate for usewith CT.

Also provided herein is a method of locating fibrin in a mammal, themethod comprising: a) administering to the mammal an imaging agent asdescribed herein; b) acquiring a first and second image of the mammalusing two imaging methods selected from 1) nuclear imaging technique, 2)magnetic resonance imaging, 3) computed tomography, and 4) opticalimaging (e.g., near infrared imaging), wherein the selected imagingmethods are appropriate for the imaging reporters present in the imagingagent of step a). In some embodiments, when optical imaging is used toimage the mammal, it is used to obtain the second image. In some cases,the first and second images are acquired simultaneously.

Further provided herein is a kit that includes an imaging agent asdescribed herein.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates phantoms imaged at 1.5 T. Top row are axial slicescorresponding to the point indicated by the line through the coronalimages (bottom row). A) pure water; B) 5 mg/mL fibrinogen; C)[Gd(HP-DO3A)] at 25 μM in 5 mg/mL fibrinogen; D) [Gd(HP-DO3A)] at 25 μMin 5 mg/mL fibrin gel; E) GdDOTA-cyclic-FBP-p-XD-FITC (compound 17) at25 μM in 5 mg/mL fibrinogen; F) GdDOTA-cyclic-FBP-p-XD-FITC (compound17) at 25 μM in 5 mg/mL fibrin gel.

FIG. 2 shows phantoms imaged with PET. Activity in each sample was 150nCi. At left are coronal images corresponding to the point indicated bythe lines through the axial images (right hand side). A) ⁶⁴CuDOTA infibrinogen; B) ⁶⁴CuDOTA in fibrin gel; C)FITC-hexyl-cyclic-FBP-p-XD-⁶⁴CuDOTA (compound 12) in fibrinogen; D)FITC-hexyl-cyclic-FBP-p-XD-⁶⁴CuDOTA (compound 12) in fibrin gel E)⁶⁴CuDOTA-cyclic-FBP-p-XD-FITC (compound 18) in fibrinogen; F)⁶⁴CuDOTA-cyclic-FBP-p-XD-FITC (compound 18) in fibrin gel.

FIG. 3 illustrates phantoms imaged using fluorescence. Top row is aphotograph of the 4 phantoms. Bottom row is the fluorescence signaloverlaid on the photographic image. A) 50 nM GdDOTA-cyclic-FBP-p-XD-FITC(compound 17) in 2 mg/mL fibrinogen solution; B) 50 nMGdDOTA-cyclic-FBP-p-XD-FITC (compound 17) in 2 mg/mL fibrin gel; C) 50nM fluorescein in 2 mg/mL fibrin gel D) 50 nM fluorescein in 2 mg/mLfibrinogen solution.

FIG. 4 shows images at the top that are orthogonal reformats of thecorresponding image immediately below it. Left: rat with occlusivethrombus in right internal carotid artery (ica). Time of flightangiography shows a flow deficit on the right side indicated by thearrows. Flowing blood appears bright in this image. Right: Black bloodT1-weighted MRI post injection of dual probe 21. The region that showedno flow on the angiographic image is now bright indicating the presenceof thrombus.

FIG. 5 illustrates orthogonal multiplanar co-registered images coveringthe MR field of view. A) PET images reconstructed from data 30-90 minpost probe; B) T1-weighted black blood MR images post probe; C)T1-weighted black blood MR images pre-probe. Arrows denote increased PETsignal (A) that is hyperintense on MR post probe (B) but not on MRpre-probe (C) and which corresponds to thrombus in the right

FIG. 6 shows time activity curves after injection of dual probe 21 intoa rat with occlusive thrombus in the right ica. Time-activity curves forthe thrombus (filled circles) and contralateral (left carotid) vessel(open circles) show rapid and static uptake in the thrombus while in thecontralateral vessel activity is clearing with time.

FIG. 7 illustrates a hyperintense PET signal in the right internalcarotid (ICA) after intravenous injection of dual probe 21 into a rabbitwith occlusive thrombus in the right ICA. From top to bottom: threeorthogonal views (axial, coronal, sagittal) of a rabbit neck. Left: MRangiogram acquired after injection of blood pool MR contrast agentgadofosveset. Contrast agent makes arteries and veins appear bright.Note signal void in right internal carotid artery (arrows). Middle: PETpost ⁶⁴Cu-labeled compound 21. Note the region of high activity; Right:merged MR-PET image shows localization of hyperintense PET signal toarea of right internal carotid artery (ICA) occlusion.

DETAILED DESCRIPTION

This disclosure provides peptide-targeted imaging probes that contain atleast two imaging reporters. An imaging reporter (IR) is a group thatmakes the probe visible in a particular imaging modality. For instance,an IR can be a complex of Gd(III) to make the probe detectable by MRI,it can be a radionuclide to make the probe detectable by PET or SPECTimaging, or it can be a fluorophore detectable by optical imaging. Thetwo or more imaging reporters can be the same or can be different. Ininstances where the imaging reporters differ, it can be possible todetect the probe using two different imaging modalities. Surprisingly,appending different imaging reporters to a short fibrin specific peptidedid not affect the affinity of the resultant probe to fibrin.

The peptide-targeted imaging probes can be used in a variety of ways toimage fibrin. For example, a fibrin-specific imaging agent that containsa radioactive element can be prepared. After administering thisradio-labeled fibrin-specific agent, an image can be obtained using anuclear imaging technique like PET or SPECT that detects the imagingagent directly. A second image can then be obtained to acquire a highresolution anatomical map using either MRI or CT. The images can then beoverlaid to localize the low resolution fibrin-targeted image within thehigh resolution anatomical image.

Another embodiment involves synthesis of a fibrin-specific imaging agentthat contains two separate imaging reporters: e.g. a fluorescent dye anda radioactive element, a fluorescent dye and a MR active imaging moiety,or a radioactive element and a MR active imaging moiety. Afteradministering this fibrin-specific agent, two images are obtained usingthe modalities appropriate to the imaging reporter, e.g. PET andoptical.

Fibrin-Specific Imaging Agents

An imaging agent, as provided herein, incorporates a fibrin bindingpeptide to allow for specific imaging of fibrin (e.g., thrombi, solidtumors, and atherosclerotic plaques) within a mammal. Any peptidecapable of binding fibrin may be used. For example, the peptidesdisclosed in WO 2008/071679, U.S. Pat. Nos. 6,984,373; 6,991,775; and7,238,341 and U.S. Patent Application No. 2005/0261472 may be used. Apeptide can be from about 2 to about 25 amino acids in length (e.g.,about 3 to about 20, about 5 to about 18, about 8 to about 15, and about10 to about 14).

A fibrin binding peptide can have the general formula:

wherein X₁ is selected from the group consisting of:

wherein X₂ is selected from the group consisting of:

X₃ is selected from the group consisting of H and OH; X₄ is selectedfrom the group consisting of H, I, Br, and Cl; X₅ is selected from thegroup consisting of H and CH₂COOH;X₆ is selected from the group consisting of:

X₇ is selected from the group consisting of CH₂CH₂C(O)NH₂ andCH₂CH(CH₃)_(2.)

In some embodiments, the fibrin binding peptide can comprise:

The ability of peptides to bind fibrin can be assessed by knownmethodology. For example, affinity of the peptide for fibrin can beassessed using the DD(E) fragment of fibrin, which contains subunits of55 kD (Fragment E) and 190 kD (Fragment DD). The DD(E) fragment can bebiotinylated and immobilized via avidin to a solid substrate (e.g., amulti-well plate). Peptides can be incubated with the immobilized DD(E)fragment in a suitable buffer and biding detected using knownmethodology. See, for example, WO 2001/09188.

Binding can also be assessed in a blood plasma-derived clot assay assay(see e.g. Overoye-Chan et al. J Am Chem Soc 2008 130:6025-39). Here,known concentrations of peptide are incubated in blood plasma (human orother species), and thrombin is added to induce clot formation. The clotis separated from the serum, and the concentration of the peptide in theserum ([peptide]free) is determined (e.g. by HPLC or if the peptide islabeled with a fluorophore by fluorescence, or if labeled by aradionuclide concentration is determined by radioactivity). Theconcentration of fibrin-bound peptide ([peptide]bound) is calculated bysubtraction ([peptide]bound)=[peptide]total −[peptide]free).

Binding can also be assessed by a dried fibrin assay. Here, purifiedfibrinogen (2.5 mg/mL; 7 μM fibrin) is clotted with thrombin and driedto a thin film in wells of a microtiter plate. The resulting clots bindto the plate without loss of protein. The clots are rehydrated withbuffer containing known concentrations of peptide. After incubation at37° C. for 2 hr, the concentration of peptide in the supernatant([peptide]free) is determined (e.g. by HPLC or if the peptide is labeledwith a fluorophore by fluorescence, or if labeled by a radionuclideconcentration is determined by radioactivity). The concentration offibrin-bound peptide ([peptide]bound) is calculated by subtraction([peptide]bound)=[peptide]total−[peptide]free). A dissociation constant(Kd) for fibrin binding can be determined by fitting a plot of[peptide]bound versus [peptide]free to either a stoichiometric (see e.g.Nair et al., Angew Chem Int Ed 2008 47:4918-21) or equivalent bindingsites model (see e.g. Overoye-Chan et al. J Am Chem Soc 2008130:6025-39).

Peptides may be synthesized directly using conventional techniques,including solid-phase peptide synthesis, solution-phase synthesis, etc.See, for example, Stewart et al., Solid-Phase peptide Synthesis (1989),W.H. Freeman Co., San Francisco; Merrifield, J. Am. Chem. Soc., 196385:2149-2145; Bodanszky and Bodanszky, The Practice of Peptide Synthesis(1984), Springer-Verlag, New York. Peptides may also be prepared orpurchased commercially. Automated peptide synthesis machines, such asmanufactured by Perkin-Elmer Applied Biosystems, may also be used.

The fibrin binding peptide is preferably purified once it has beenisolated or synthesized by either chemical or recombinant techniques.For purification purposes, there are many standard methods that may beemployed including reversed-phase high-pressure liquid chromatography(RP-HPLC) using an alkylated silica column such as C₄-, C₈- orC₁₈-silica. A gradient mobile phase of increasing organic content isgenerally used to achieve purification, for example, acetonitrile in anaqueous buffer, usually containing a small amount of trifluoroaceticacid. Ion-exchange chromatography can also be used to separate peptidesbased on their charge. The degree of purity of the fibrin bindingpeptide may be determined by various methods, including identificationof a major large peak on HPLC. A peptide that produces a single peakthat is at least 95% of the input material on an HPLC column ispreferred. Even more preferable is a peptide that produces a single peakthat is at least 97%, at least 98%, at least 99% or even 99.5% of theinput material on an HPLC column.

To facilitate imaging of the fibrin, the fibrin binding peptide isdetectably labeled with two or more imaging reporters. Each imagingreporter can be independently selected from a chelator comprising aradioactive metal ion, a chelator comprising a paramagnetic metal ion,and a fluorescent dye. In some embodiments, the two or more imagingreporters are the same.

A chelator is a polydentate ligand which is capable of coordinating aradioactive or paramagnetic metal ion. Suitable chelators are known inthe art and include acids with methylene phosphonic acid groups,methylene carbohydroxamine acid groups, carboxyethylidene groups, orcarboxymethylene groups. Examples of chelators include, but are notlimited to, diethylenetriaminepentaacetic acid (DTPA),1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetraacetic acid (DOTA),ethylenediaminetetraacetic acid (EDTA), and1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA).Additional chelating ligands are ethylenebis-(2-hydroxy-phenylglycine)(EHPG), and derivatives thereof, including 5-Cl-EHPG, 5Br-EHPG,5-Me-EHPG, 5t-Bu-EHPG, and 5 sec-Bu-EHPG; benzodiethylenetriaminepentaacetic acid (benzo-DTPA) and derivatives thereof, includingdibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl-DTPA, and dibenzylDTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) andderivatives thereof, the class of macrocyclic compounds which contain atleast 3 carbon atoms, more preferably at least 6, and at least twoheteroatoms (O and/or N), which macrocyclic compounds can consist of onering, or two or three rings joined together at the hetero ring elements,e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is1,4,7-triazacyclononane N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA,where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyltetraacetic acid), and benzo-TETMA, where TETMA is1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid);derivatives of 1,3-propylenediaminetetraacetic acid (PDTA) andtriethylenetetraaminehexaacetic acid (TTHA); derivatives of1,5,10-N,N′,N″-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM) and1,3,5-N,N′,N″-tris(2,3-dihydroxybenzoyl) aminomethylbenzene (MECAM).Additional examples of representative chelators and chelating groups aredescribed in WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/20182, U.S. Pat. Nos.4,899,755 and 6,991,775, and U.S. Patent Application No. 2005/0261472.

In some embodiments, a chelator can be, for example, linear,macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S.Pat. Nos. 5,367,080, 5,364,613, 5,021,556, 5,075,099, 5,886,142), andother chelators known in the art including, but not limited to, HYNIC,DTPA, EDTA, DOTA, TETA, and bisamino bisthiol (BAT) chelators (see alsoU.S. Pat. No. 5,720,934). The chelates may be covalently linked directlyto the fibrin binding moiety or linked to the fibrin binding moiety viaa linker, as described below, and then directly labeled with the metalion (e.g., paramagnetic or radioactive ion) of choice (see, WO 98/52618,WO 2008/071679, U.S. Pat. Nos. 5,879,658, and 5,849,261).

In some embodiments, a chelator is selected from the group consistingof:

wherein M is a radioactive metal ion or a paramagnetic metal ion. Insome embodiments, the chelator is:

In some cases, a fibrin binding peptide can be labeled with two imagingreporters, wherein the two imaging reporters are selected from the pairsconsisting of: a fluorescent dye and a chelator comprising a radioactivemetal ion; a fluorescent dye and a chelator comprising a paramagneticmetal ion; and a chelator comprising a radioactive metal ion and achelator comprising a paramagnetic metal ion.

In some embodiments, a fibrin binding peptide can be labeled with threeimaging reporters, wherein two of the imaging reporters are selectedfrom the pairs consisting of a fluorescent dye and a chelator comprisinga radioactive metal ion; fluorescent dye and a chelator comprising aparamagnetic metal ion; and a chelator comprising a radioactive metalion and a chelator comprising a paramagnetic metal ion. In someembodiments, a fibring binding peptide can be labeled with four imagingreporters, wherein two of the imaging reporters are selected from thepairs consisting of a fluorescent dye and a chelator comprising aradioactive metal ion; fluorescent dye and a chelator comprising aparamagnetic metal ion; and a chelator comprising a radioactive metalion and a chelator comprising a paramagnetic metal ion. In suchembodiments with 3 or 4 imaging reporters, the remaining imagingreporter(s) can be selected from any described herein.

The fibrin binding peptide may be conjugated with an imaging reporteragent comprising a chelator comprising a radioactive metal ionappropriate for single photon emission computed tomography (SPECT)and/or positron emission tomography (PET) imaging. A radioactive metalion can be selected from ⁴⁵Ti, ⁵¹Mn, ⁵²Mn, ^(52m)Mn ⁵²Fe, ⁶⁰Cu, ⁶¹Cu,⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ⁸⁶Y, ⁸⁹Zr, ^(94m)Tc, ^(99m)Tc, ¹¹⁰In,¹¹¹In, ¹¹³In, ¹⁷⁷Lu.

In some embodiments, at least one of the radioactive metal ions is ⁶⁴Cu.Copper-64 (t_(1/2)=12.7 h; E(β⁺)^(max)=656 keV; E(β⁻)^(max)=573 keV) isa useful radionuclide for PET applications. The longer half-life of ⁶⁴Curelative to other positron emitting isotopes like ¹¹C (20 min) means noonsite cyclotron is required for production. Thus ⁶⁴Cu can be shipped tousers all over the country. An additional benefit to ⁶⁴Cu is that the⁶⁴Cu label is introduced in the ultimate synthetic step via a highlythermodynamically favored chelation reaction which leads to higherspecific activity and potentially no requirement for final HPLCpurification. The long half-life and potential ease of preparation meanthat Cu-64 based molecular imaging probes have the potential to be morewidely available to the nuclear medicine community, either in a kit formwhere the end user mixes the ⁶⁴Cu with a probe precursor to formulatethe probe or if the formulated probe is delivered by a centralizedsupplier. ⁶⁴Cu is a particularly useful isotope for fibrin imaging. Inhuman clinical trials with a fibrin-targeted MR probe it was found thatimaging was best 2 to 24 hrs post injection of the probe (see, e.g.,Vymazal J. et al., Invest Radiol. 2009 44(11):697-704; and Spuentrup E.et al., Eur Radiol. 2008 18(9): 1995-2005). For a PET application it ispreferred to use a radionuclide whose half-life is similar to thebiological time scale.

A paramagnetic metal ion can be conjugated to a fibrin binding peptidethrough a chelating group. Examples of suitable paramagnetic metal ionsinclude, but are not limited to, those metal ions having atomic numbers21-29, 42, 44, or 57-83. This includes ions of the transition metal orlanthanide series which have at least one (e.g., at least two, at leastfour, at least five) unpaired electrons and a magnetic moment of atleast 1.7 Bohr magneton. In some embodiments, a paramagnetic metal ionis selected from the group consisting of Gd(III), Fe(III), Mn(II andIII), Cr(III), Cu(II), Dy(III), Tb(III), Ho(III), Er(III), and Eu(III).For example, a paramagnetic metal ion can be Gd(III), Mn(II), orMn(III).

A fibrin binding peptide can also be conjugated to a fluorescent dye.Examples of fluorescent dyes include, but are not limited to,fluorescein and derivatives thereof (e.g., fluorescein isothiocyanate(FITC)), Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS, rhodamine 6G(R6G) and derivatives thereof (e.g., tetramethylrhodamine (TMR),tetramethylrhodamine isothiocynate (TMRITC), and x-rhodamine), Texasred, BODIPY FL (Molecular Probes Corporation, U.S.A.), BODIPY FL/C3,BODIPY FL/C6, BODIPY 5-FAM, BODIPY TMR, and their derivatives (e.g.,BODIPY TR, BODIPY R6G, and BODIPY 564) Dapoxyl® dyes, PyMPO derivatives(PyMPO maleimide, PyMPO-OSu), Prodan and derivatives (BADAN, Acrylodan),Dansyl derivatives (IAEDANS, Dansyl chloride), NDB derivatives (NBD-Cl,IANBD), Coumarin and derivatives (MDCC, DACIA, DACITC, CPM), Merocyaninederivatives (Merocyanine 540), amd Dimethylaminophthalimides (4-DMAP,4-DMN, 6-DMN).

In some embodiments, a fluorescent dye is selected from the groupconsisting of:

wherein:each m is independently an integer from 0 to 6; andX is selected from the group consisting of SO₃ ⁻, OH, PO₄ ²⁻.

The imaging reporters described herein may be directly bound to thefibrin binding peptide or conjugated through a linker moiety. A linkercan be used to covalently attach one or more imaging reporters to thepeptide terminus. The linker may be branched or unbranched and maycomprise multiple functional groups for imaging reporter attachment.Linkers, if present, typically are relatively small and rigid for theimaging agents described herein. For example, a linker can have amolecular weight less than about 350 (e.g., less than about 200).

In some embodiments, a linker is independently selected from the groupconsisting of:

—NHCH(R)C(O)—, wherein R is any natural amino acid side chain;

—NH(CH₂)_(n)C(O)—, wherein n is an integer from 1-6;

—NHCH₂CH₂OCH₂CH₂C(O)—;

—NHCH₂CH₂OCH₂CH₂OCH₂CH₂C(O)—;

—NHCH₂C₆H₄CH₂NH—;

—NH(CH₂)_(m)NH—, wherein m is an integer from 2-6;

—NHCH₂OCH₂NH—;

—NHCH₂CH₂OCH₂CH₂NH—; and

—NHCH₂CH₂OCH₂CH₂OCH₂CH₂NH—.

In some cases, a fibrin-specific imaging agent comprises a fibrinbinding peptide bound optionally through one or more linkers to two ormore imaging reporters. In some embodiments, at least one of the imagingreporters comprises ⁶⁴Cu. Examples of imaging agents include:

or a pharmaceutically acceptable salt form thereof.

In some embodiments, a fibrin-specific imaging agent can have thestructure:

In some embodiments, a fibrin-specific imaging agent can have theformula:

IR₁-L₁-FTP-L₂-IR₂

wherein:FTP is a fibrin binding peptide;L₁ and L₂ are optional linkers;IR₁ and IR₂ are imaging reporters wherein the two imaging reporters areselected from the pairs consisting of: a fluorescent dye and a chelatorcomprising a radioactive metal ion; a fluorescent dye and a chelatorcomprising a paramagnetic metal ion; and a chelator comprising aradioactive metal ion and a chelator comprising a paramagnetic metalion.

The imaging agent can be a compound of the formula:

wherein:X₁ is selected from the group consisting of:

X₂ is selected from the group consisting of:

X₃ is selected from the group consisting of H and OH;X₄ is selected from the group consisting of H, I, Br, and Cl;X₅ is selected from the group consisting of H and CH₂COOH;X₆ is selected from the group consisting of:

andX₇ is selected from the group consisting of CH₂CH₂C(O)NH₂ andCH₂CH(CH₃)₂.

When L₁ is present, it can be selected from the group consisting of:

—NHCH(R)C(O)—, wherein R is any natural amino acid side chain;

—NH(CH₂)_(n)C(O)—, wherein n is an integer from 1-6;

—NHCH₂CH₂OCH₂CH₂C(O)—; and

—NHCH₂CH₂OCH₂CH₂OCH₂CH₂C(O)—.

L₂, when present, can be selected from the group consisting of:

—NHCH₂C₆H₄CH₂NH—;

—NH(CH₂)_(m)NH—, wherein m is an integer from 2-6;

—NHCH₂OCH₂NH—;

—NHCH₂CH₂OCH₂CH₂NH—; and

—NHCH₂CH₂OCH₂CH₂OCH₂CH₂NH—.

In some embodiments, at least one of IR₁ and IR₂ is a chelatorcomprising ⁶⁴Cu.

Examples of an imaging agent comprising a compound of the formuladescribed above include:

wherein M is a radioactive or paramagnetic metal ion.

Also provided herein is a fibrin-specific imaging agent having thestructure:

or a pharmaceutically acceptable salt form thereof, wherein each M isindependently a radioactive metal ion or Gd³⁺, provided that no morethan three M are Gd³⁺. In some embodiments, at least one M is selectedfrom the group consisting of: ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶y, ⁸⁹Zr,^(99m)Tc, and ¹¹¹In. In some embodiments, three M are Gd³⁺ and one M isa radioactive metal ion. In another embodiment, two M are Gd³⁺ and theremaining two Ms are independently a radioactive metal ion. In a furtherembodiment, one M is Gd³⁺ and the remaining three Ms are independently aradioactive metal ion. In some embodiments, where multiple Ms are aradioactive metal ion, the Ms can be the same. In some embodiments, theimaging agent may require one or more countercations (e.g., sodium,potassium, ammonium).

The fibrin-specific imaging agents described herein can be preparedusing conventional synthetic methods known to those of skill in the art.See, for example, U.S. Pat. Nos. 6,984,373; 6,991,775; and 7,238,341 andU.S. Patent Application No. 2005/0261472, as well as the Examplesdetailed below. The specific parameters included in the examples areintended to illustrate and are not presented to in any way limit thedisclosure.

Imaging agents employing metallo-radionuclides are often prepared bychelating the radionuclide in the final synthetic step. It is useful toavoid HPLC purification of the final product as this purification stepis time consuming, involves organic solvents which must be removed fromthe purified product prior to use, and generally results in lower yieldsbecause of radioactive decay during the purification process. Thefibrin-specific imaging agents described herein incorporate potentchelators to quantitatively bind the radionuclide often avoiding theneed for any additional purification. If there is any radionuclidepresent that is not the final product, then this can be easily removedin two ways based on ion exchange chromatography. Themetallo-radionuclide precursors are typically cationic species, e.g.⁶⁴Cu²⁺, ⁶⁸Ga³⁺. If analytical radio-HPLC or radio-TLC indicate that theimpurity is unreacted metallo-radionuclide, then the reaction mixturecan be passed through a cationic exchange column, e.g. Dowex-50 resin,sodium form. The unreacted cationic radionuclide will adhere to thecolumn while the desired negatively charged or neutral imaging agentwill pass through the column unchanged. This is a fast purification thatuses no organic solvent and results in pure product. An alternate methodis to add cation exchange resin, e.g. Dowex-50 resin, sodium form,directly to the solution to bind the cationic radionuclide impurity. Thesolution is then filtered to remove the resin containing the impurity.

It is also possible that other impurities may be formed. For instance itis possible for the metallo-radionuclide to bind weakly to amino acidside chains of the peptide and not be bound by the chelator. This may beencountered when preparing imaging agents of very high specificactivity. In those instances, a positively charged weak chelator can beadded in excess to scavenge radionuclide weakly associated with thepeptide. For instance diethylenetriamine (dien) exists in solution at pH7 as a di-cation, H₂dien^(2+.) Diethylenetriamine forms stable complexeswith the Cu²⁺ ion resulting in a positively charged complex, Cu(dien)²⁺.However dien is not capable of displacing Cu²⁺ from the more stablechelators used in this application, e.g. DOTA, NOTA, CB-TE2A, etc. Afterincubating the reaction mixture briefly with diethylenetriamine, theresultant solution is passed through a cation exchange column orcartridge, e.g. Dowex-50 resin, sodium form. The cation exchange columnremoves the positively charged H₂dien²⁺ and Cu(dien)²⁺ but the desirednegatively charged or neutral imaging agent will pass through the columnunchanged. Alternately, the cation exchange resin can be directly addedto the solution, and then filtered to remove the resin containing theimpurity. Again, these purification methods are fast and avoid the useof organic solvents.

One skilled in the art can appreciate that other weak chelating ligandsthat form cationic complexes with the specific radionuclide used couldbe employed. For instance, the reference Critical Stability Constants,vols. 1-6 edited by A. E. Martell and R. M. Smith, Plenum Press, 1989could be consulted to find appropriate ligands for an appropriate metalion. Similarly, other cation exchange resins could be used.

Methods of Use

The fibrin-specific imaging agents described herein can be used to imagefibrin present in a mammal. For example, the imaging agents can be usedto image thrombi, solid tumors, and atherosclerotic plaques. Anysuitable method of imaging fibrin, as appropriate for the imagingreporters present on the imaging agent, may be used.

For example, provided herein is a method of imaging fibrin in a mammal.In some cases, the method can include:

-   -   a) administering to the mammal an imaging agent as described        herein, wherein the imaging agent comprises at least one imaging        reporter comprising a chelator and a radioactive metal ion;    -   b) acquiring an image of the mammal using a nuclear imaging        technique;    -   c) acquiring an image of the mammal using magnetic resonance        imaging or computed tomography; and    -   d) overlaying the images of steps b) and c) to image the fibrin        within the mammal.

A nuclear imaging technique, as described herein, can include SPECT andPET. Without being bound by theory, the nuclear imaging technique can beused to detect and image the imaging agent directly. The second imagecan then be used to acquire a high resolution anatomical map using, forexample, magnetic resonance imaging (MRI) or computed tomography (CT).The images are then overlaid or fused to localize the low resolutionfibrin-targeted image within the high resolution anatomical image. Forexample, to identify the presence of thrombus in a specific bloodvessel, a CT angiogram can then be obtained and this image fused withthe nuclear medicine image. The CT image would show the vascular treeand the PET or SPECT image would identify the thrombus in a specificartery or vein. The order of acquiring the images is unimportant: the MRor CT image can be acquired prior to, after or simultaneous with thenuclear image.

Overlaying of images can be done by various means known in the art. See,for example, U.S. Pat. Nos. 7,412,279; 7,110,616; 6,898,331; 6,549,798;and 5,672,877; Rudd, J. H F. et al., J. Nucl. Med. 2008 49(6): 871-878;Slomka, P. J. et al., J. Nucl. Med. 2009 50: 1621-1630; and Jupp, B. andO'Brien, T. J., Epilepsia 2007 49: 82-89. In some embodiments, the firstand second image data sets can be overlaid to determine the presence ofthe fibrin within the mammal, provided that the image acquired usingnuclear imaging indicated the presence of fibrin. For example, the firstand second image data sets can be combined to produce a third data setthat includes an image of the fibrin target and an image of anatomicalregion where the fibrin is located. The third data set is capable ofindicating the location of the fibrin, if present, within the mammal. Ifdesired, the third data set may be displayed on a display device inorder to indicate the location of the stationary target within thevascular system. The third data set may also indicate the size of thestationary target within the mammal.

In some embodiments, the MR or CT imaging can employ a contrast agent,wherein the contrast agent is non-specific for fibrin or thrombi. Such acontrast agent can be any known to those of skill in the art. Forexample, Gd(HP-DO3A), Gd(DTPA), Gd(BOPTA), Gd(EOB-DTPA), Gd(DOTA),Gd(DTPA-BMA), gadoversetamide, gadofosveset for use with MRI oriopamidol, iohexol, ioxilan, iopromide, iodixanol, ioxaglate,metrizoate, or diatrizoate for use with CT.

Further provided herein is a method of imaging fibrin using an imagingagent having at least two imaging reporters, wherein the at least twoimaging reporters are selected from the pairs consisting of: afluorescent dye and a chelator comprising a radioactive metal ion; afluorescent dye and a chelator comprising a paramagnetic metal ion; anda chelator comprising a radioactive metal ion and a chelator comprisinga paramagnetic metal ion. The method includes:

-   -   a) administering to a mammal the imaging agent described above;        and    -   b) acquiring a first and second image of the mammal using two        imaging methods selected from 1) nuclear imaging technique, 2)        magnetic resonance imaging, 3) computed tomography, and 4)        optical imaging, wherein the selected imaging methods are        appropriate for the imaging reporters present in the imaging        agent of step a).

Optical imaging, as described herein, can include near infrared (NIR)imaging. The two images may be acquired sequentially or simultaneously.In some cases it may be beneficial to acquire an image with the lowerresolution modality first and then use this image to direct where thesecond higher resolution image is acquired. For example, a non-invasiveimage can be obtained using a nuclear imaging technique like PET orSPECT (if radiolabeled) or MRI (if labeled with gadolinium) that issensitive to the imaging agent. This first, non-invasive image is usedto identify the region for invasive NIR imaging. A NIR image can then beobtained, e.g. using a fiber optic catheter, and a thrombus on thecoronary vessel wall can be imaged for the purpose of guiding surgicaltherapy. Alternately a NIR image may be obtained in the surgical fieldto guide the surgeon to the margins of a tumor.

The imaging agents and methods described herein can be used in numerousdiagnostic and therapeutic applications. For example, imaging thrombi ina mammal, detecting the presence or absence of thrombi in a mammal,imaging a solid tumor in a mammal detecting the presence or absence of asolid tumor in a mammal, imaging an atherosclerotic plaque in a mammal,detecting the presence or absence of an atherosclerotic plaque in amammal.

Pharmaceutical Compositions and Salts

Pharmaceutical compositions as described herein comprise at least onefibrin-specific imaging agent, or pharmaceutically acceptable saltsthereof, and one or more pharmaceutically acceptable ingredients,excipients, carriers, adjuvants and/or vehicles.

Pharmaceutical compositions can be administered to mammals includinghumans in a manner similar to other diagnostic or therapeutic agents.The dosage to be administered and the mode of administration will dependon a variety of factors including age, weight, sex, condition of thepatient, and genetic factors, and will ultimately be decided by theattending physician or veterinarian. In general, dosage required fordiagnostic sensitivity or therapeutic efficacy will range from about0.001 to 50,000 g/kg, more usually 0.01 to 25.0 g/kg of host body mass.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins,such as human serum albumin, buffer substances such as phosphates,glycine, sorbic acid, potassium sorbate, partial glyceride mixtures ofsaturated vegetable fatty acids, water, salts or electrolytes, such asprotamine sulfate, disodium hydrogen phosphate, potassium hydrogenphosphate, sodium chloride, zinc salts, colloidal silica, magnesiumtrisilicate, polyvinyl pyrrolidone, cellulose-based substances,polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycoland wool fat.

The pharmaceutical compositions may be administered by a variety ofroutes or modes. These include, but not limited, to oral, intratracheal,sublingual, pulmonary, topical, rectal, nasal, buccal, vaginal,parenteral, or via an implanted reservoir. Implanted reservoirs mayfunction by mechanical, osmotic, or other means. The term parenteral asused herein includes intraperitoneal, paravertebral, periarticular,periostal, subcutaneous, intracutaneous, intravenous, intra-arterial,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques.

Such compositions are preferably formulated for parenteraladministration, and most preferably for intravenous or intra-arterialadministration. Generally, and particularly when administration isintravenous or intra-arterial, pharmaceutical compositions may be givenas a bolus, as two or more doses separated in time, or as a constant ornonlinear flow infusion.

Details concerning dosages, dosage forms, modes of administration,composition and the like are further discussed in a standardpharmaceutical text, such as Remington's Pharmaceutical Sciences, 18thed., Alfonso R. Gennaro, ed. (Mack Publishing Co., Easton, Pa. 1990),which is hereby incorporated by reference.

The imaging agents described herein can also be in the form of apharmaceutically acceptable salt. The term “pharmaceutically acceptablesalt,” as used herein, refers to derivatives of the imaging agentswherein the parent compound is modified by making the acid or basicgroups not yet internally neutralized in the form of non-toxic, stablesalts which does not destroy the pharmacological activity of the parentcompound.

Suitable examples of the salts include: mineral or organic acid salts,of basic residues such as amines; alkali or organic salts of acidicresidues such as carboxylic acids; and the like.

Preferred cations of inorganic bases which can be suitably used toprepare salts comprise ions of alkali or alkaline-earth metals such aspotassium, sodium, calcium or magnesium. Preferred cations of organicbases comprise, inter alia, those of primary, secondary and tertiaryamines such as ethanolamine, diethanolamine, morpholine, glucamine,N-methylglucamine, N,N-dimethylglucamine.

Preferred anions of inorganic acids which can be suitably used to salifythe imaging agents include the ions of halo acids such as chlorides,bromides, iodides or other ions such as sulfate. Preferred anions oforganic acids comprise those of the acids routinely used inpharmaceutical techniques for the salification of basic substances suchas, for instance, acetate, succinate, citrate, fumarate, maleate oroxalate.

Preferred cations and anions of amino acids comprise, for example, thoseof taurine, glycine, lysine, arginine, ornithine or of aspartic andglutamic acids.

Pharmaceutical compositions can also include stabilizers and/orstabilizer combinations that slow or prevent radiolytic damage toimaging agents. Any stabilizer known to inhibit radiolytic damage toradiolabeled compounds may be used alone or in combination with otherstabilizers. For example, human serum albumin (HSA), ascorbate, phenol,sulfites, glutathione, cysteine, gentisic acid, nicotinic acid, ascorbylpalmitate, PO₂H₃, glycerol, sodium formaldehyde sulfoxylate, Na₂S₂O₅,Na₂S₂O₃, SO₂, and mixtures thereof. See also, U.S. Patent ApplicationNo. 2007/0269375 and WO 1995/025119.

Kits

Also provided herein are kits. Typically, a kit includes one or morefibrin-specific imaging agents as described herein. In certainembodiments, a kit can include one or more delivery systems for theimaging agent, and directions for use of the kit (e.g., instructions fortreating a subject).

In some embodiments, a kit can include one or more imaging agentprecursors, wherein the imaging agent has not been chelated to aparamagnetic or radioactive metal ion (e.g., ⁶⁴Cu). Such precursors maybe present in the form of a lyophilized powder or as a sterile solution.In such cases, the kit can further include a labile form of the metalion (e.g., an acetate or halide salt) and/or directions for use of thekit (e.g., instructions for the final chelation of the metal ion to theimaging agent prior to administration to a mammal).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this disclosure pertains. All patents, applications,published applications, and other publications are incorporated byreference in their entirety. In the event that there is a plurality ofdefinitions for a term herein, those in this section prevail unlessstated otherwise.

EXAMPLES Example 1 Cu-64 Bifunctional Chelator

Eisenwiener (Eisenwiener K P, Prata M I, Buschmann I, et al. BioconjugChem. 2002 13(3):530-41) described the synthesis of racemic NODAGAstarting from protected α-bromoglutaric acid. The optically pure isomeris preferred since this will result in a single isomer when coupled to apeptide, rather than diasteromers if the racemate is used. The mesylateof the differentially protected, enantiomerically pureS-2-hydroxy-1,5-pentanedioic acid derivative has been described (U.S.Application No. 2007/0244316). Reacting this with 2 equivalents ofcommercial triazacyclononane (TACN) gives the monosubstituted product.Excess TACN is removed by washing with water. Alkylation of theremaining nitrogens with tert-butyl protected bromoacetic acid gives theprotected R-NODAGA intermediate. Removal of the benzyl group byhydrogenation gives the advanced bifunctional intermediate product shownfollowing step (iii) in Scheme 1. This molecule can then be coupled toamino groups on peptides via the free acid. Acid deprotection of thetert-butyl groups followed by chelation with copper gives the desiredproduct.

Example 2 Fibrin-Specific PET Probes

A fibrin-specific PET probe is described in Scheme 2 and prepared asfollows. First p-xylenediamine is coupled to 2-chlorotritylchlorideresin in DMF using diisopropylethylamine (DIPEA) as a base as describedpreviously (Overoye-Chan K, Koerner S, Looby R J, et al. J Am Chem Soc.2008 130(18):6025-39). Standard solid phase peptide coupling is thenused to elongate the peptide. The peptide used in this example is thesame as used in EP-2104R (Overoye-Chan K, Koerner S, Looby R J, et al. JAm Chem Soc. 2008 130(18):6025-39). The completed peptide is thencleaved from the resin and deprotected using trifluoroacetic acid. Thepeptide is precipitated by addition of cold diethyl ether. This linearpeptide is then taken up in a 1:1 mixture of DMSO and pH 4.5 sodiumacetate buffer and allowed to cyclize overnight via DMSO oxidation. Atthis stage the resultant peptide diamine may be purified by HPLC. Thispeptide is then reacted with two equivalents of NODAGA t-butyl ester(see Example 1) using standard coupling conditions of HOBT and PyBOP inDMF. The tert-butyl groups are then removed using TFA and the productprecipitated with cold ether. This precursor is then taken up in sodiumacetate buffer, pH 5.5, and reacted with 20 mCi of ⁶⁴CuCl₂ for 60minutes at 60° C. The solution is then passed through a cation exchangecartridge (Na⁺ form) and then through a 0.2 μm filter into a sterilevial.

Example 3 Synthesis Dual PET-NIR Fibrin-Specific Probes

Starting from the protected peptide still on resin as described inExample 2, the NODAGA t-butyl ester from Example 1 is converted to thepentafluorophenol ester (PFP) (Overoye-Chan K, Koerner S, Looby R J, etal. J Am Chem Soc. 2008 130(18):6025-39) and coupled to the N-terminus.After coupling the chelator to the resin, the peptide is cleaved fromthe resin and deprotected in a single step using a TFA (88 parts),triisopropyl silane (4), thioanisole (4), water (4) cocktail.Precipitation with cold ether gives the deprotected peptide with aprimary amine at the C-terminus (Scheme 3). The peptide is cyclizedusing DMSO as described in the Example 2. The near infrared dyeAlexaFluor 750 purchased as the succinimidyl ester is conjugated to theC-terminus in solution. HPLC purification gives the probe precursordenoted Cu-Pep-Fluor. This precursor is then taken up in sodium acetatebuffer, pH 5.5, and reacted with 20 mCi of ⁶⁴CuCl₂ for 60 minutes at 60°C. The solution is then passed through a cation exchange cartridge (Na⁺form) and then through a 0.2 μm filter into a sterile vial.

Alternatively, the dye is conjugated to the N-terminus while the peptideis still on resin. The dye conjugated peptide is cleaved from the resinand deprotected with TFA, precipitated with ether and then cyclized withDMSO. One equivalent of NODAGA-PFP is then coupled to the C-terminalamino group in solution. The t-Bu protecting groups on the NODAGA ligandare removed by treatment with TFA. HPLC purification gives the probeprecursors denoted Fluor-Pep-Cu. This precursor is then taken up insodium acetate buffer, pH 5.5, and reacted with 20 mCi of ⁶⁴CuCl₂ for 60minutes at 60° C. The solution is then passed through a cation exchangecartridge (Na⁺ form) and then through a 0.2 μm filter into a sterilevial.

Example 4 Synthesis of Dual MRI-NIR Probe

The same protected peptide on resin can be used to prepare probes with 2gadolinium chelates at the N-terminus (for MRI contrast) and a NIR dyeat the C-terminus, Scheme 4. Using Fmoc protected lysine at theN-terminus, 2 DOTAGA-PFP synthons (Overoye-Chan K, Koerner S, Looby R J,et al. J Am Chem Soc. 2008 130(18):6025-39) can be reacted with thepeptide on solid phase. The strong acid cleavage cocktail describedabove results in a deprotected peptide off resin with both DOTAGAligands deprotected and a free primary amine at the C-terminus. Thepeptide is cyclized with DMSO and then chelated by adding twoequivalents of GdCl₃ and stirring at pH 6.5 for 4 hours. Thesuccinimidyl ester of the AlexaFluor dye is then coupled in aqueoussolution. HPLC purification gives the Gd₂-Pep-Fluor construct.

Example 5 Synthesis of a Bis-Cu-64 PET Agent 1) Synthesis ofDOTAGA(OtBu)₄-OPFP (1)

DOTAGA(OtBu)₄-OH (0.4 g, 0.571 mmol, 1 eq) and pentafluorophenol (PFP,0.1262 g, 0.686 mmol, 0.361 g, 0.686 mmol, 1.2 eq) were dissolved indichloromethane (DCM). Polystyrene-carbodiimide resin (PS-DCC, 1.2 eq,1.9 mmol/g loading capacity) was added to the solution and the reactionvessel was shaken on an orbital shaker for 3 h. The reaction wasmonitored by HPLC. The reaction mixture was filtered and the solventevaporated. Yield=0.4376 g, includes unreacted PFP which does notinterfere with the next step.

2) Synthesis of the Bis(DOTAGA(OtBu)₄)-FBP-mXD (3)

The FBP-mXD (2) peptide is prepared as previously described exceptmeta-xylene was used instead of para-xylene (Overoye-Chan K, Koerner S,Looby R J, et al. J Am Chem Soc. 2008 130(18):6025-39).DOTAGA(OtBu)₄-OPFP (0.0874 g, 0.1 mmol, 1 eq.) and FBP-m-XD (0.2 g, 0.1mmol, 1 eq.) were dissolved in 5 m DMF. The pH of the solution wasincreased to 6.5 with diisopropylethylamine (DIPEA) and it was stirredfor 2 h. 0.0438 g (0.5 eq) of 1 was added and the pH was increased to6.5. Again after stirring for 30 min., another portion of 0.0438 g (0.5eq) of 1 was added and the pH was increased to 6.5. The same step wasrepeated after another 30 min. Finally, another 0.0262 g (0.3 eq) of 1was added and pH maintained at 6.5. The reaction mixture was stirred foranother hour and monitored by HPLC. A saturated brine solution was addedto the mixture and precipitation was observed. The suspension wasstirred for 30 min. The solid was filtered and dried under reducedpressure.

3) Synthesis of the Bis(DOTAGA(OH)₄)-FBP-mXD (4)

Bis(DOTAGA(OtBu)₄)-FBP-mXD was dissolved in a solution oftrifluoroacetic acid (TFA), phenol, methanesulfonic acid (MSA),thioanisole and DCM (90:2.5:2.5:2.5:2.5, ˜15 mL/g 3). The reaction wasstirred for 20 min. The solution was evaporated to a smaller volume.Diethylether was added to precipitate a solid. The solution was filteredand the solid was washed with ether. The solid was dissolved in amixture of water and acetonitrile (ACN) and injected onto a prep C4column. A gradient from 0% B (0.1% TFA/95% H₂O/5% ACN) to 100% B (0.1%TFA/10% H₂O/90% ACN) was run on the column. Starting from 0% B, thefraction of B increased to 29% over 10 minutes, then from 29 to 31% Bover 15 minutes and then from 31 to 100% B over the next 3 min. Thecolumn was washed with 100% B for 5 min and the % B was ramped to 0% inthe next 2 min. The system was re-equilibrated at 0% B over 5 minutes(total time=35 min). The peptide eluted at a concentration of 30.3% B.(M+2H)²⁺/2: Expected—1217.5, Observed—1217.6: % purity (HPLC, A₂₂₀)=85%.

4) Synthesis of Cu₂-bis-(DOTAGA(OH)₄)-FBP-mXD (5)

The Cu-64 was received in a small eppendorf tube from WashingtonUniversity in St. Louis. All the Cu-64 was transferred into a vial usinga 0.1 M NaOAc solution, pH 5.5. A 1 mM solution of peptide 4 wasprepared in 0.1 M NaOAc, pH 5.5. 50 μL of the peptide 4 solution wasadded to the Cu-64 solution and stirred at 50° C. for an hour. Thesolution was checked by RP-TLC. The free Cu-64 in the form of Cu(OAc)₂does not move with the solvent. However, the peptide labeled Cu-64 moveswith the mobile phase. If the reaction was not complete and free Cu-64was observed, more peptide was added to the reaction mixture and thereaction mixture was stirred for another 30 min at 50° C. The solutionwas checked by RP-TLC and this procedure was repeated until almost nofree Cu(OAc)₂ was left. The solution was diluted in saline beforeinjection into animals.

Example 6 Synthesis of a Bis-Gd MR Agent, Gd₂-Bis-(DOTAGA(OH)₄)-FBP-mXD(6)

Compound 4 is dissolved in water and the pH is changed to 6.5 using 1Nsodium hydroxide. Gadolinium (III) chloride hexahydrate (GdCl₃.6H₂O, 1eq) is added to the solution and the pH is adjusted to 6.5 with 1Nsodium hydroxide. The reaction is stirred for 1 h at room temperatureand monitored by HPLC. Residual gadolinium (III) chloride is neutralizedwith ethylenediaminetetraacetic acid (EDTA, 0.25 eq). The reaction isstirred for 15 min. and the pH is adjusted to 6.5 with 1N sodiumhydroxide. The neutralization is monitored by testing with xylenolorange. The product is isolated from solution and purified by reversephase chromatography.

Example 7 Synthesis of Dual MR-Optical Agent,FITC-Hexyl-Cyclic-FBP-p-XD-DOTA-Gd (11) 1) Synthesis of FBP-p-XD-SP (7)

2-Chlorotritylchloride resin (3.0069 g, 2.947 mmol, 0.98 mmol/g, 1 eq)was swollen in DCM. The resin was then washed with DMF and suspended inDMF. 1.4047 g (10.315 mmol, 3.5 eq) of p-xylylenediamine was added tothe suspension. The pH of the mixture was adjusted to 7 with DIPEA. Thereaction was agitated for 2 h. A 10 mL solution of DIPEA/DMF/MeOH(17:2:1) was added to the reaction and it was agitated for 30 min. Theresin was subsequently washed with DMF. The1,4-bisaminomethylbenzyl-2-chlorotritylchloride resin was suspended inDMF. Fmoc-Gln(Trt)-OH (6.2991 g, 10.315 mmol, 3.5 eq),diisopropylcarbodiimide (DIC, 2.282 mL, 14.735 mmol, 5.0 eq), and1-hydroxybenzotriazole (HOBT, 0.9956 g, 7.368 mmol, 2.5 eq) were added.The reaction was agitated for 4 h. The reaction was monitored by Kaiserand chloranil tests. The resin was washed with DMF (3 times) and DCM (3times). 20% piperidine in DMF was added to the resin and the reactionwas agitated for 30 min. The resin was washed with DMF (2 times), 5%HOBT and DMF (2 times). This procedure was repeated for each of thefollowing amino acids in the following order: Fmoc-Ile-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH,Fmoc-Tyr(3-Cl)—OH, Fmoc-Hyp(tBu)-OH, Fmoc-Cys(Trt)-OH,Fmoc-d-Glu(OtBu)-OH, Fmoc-Tyr(tBu)-OH.

2) Synthesis of FITC-hexyl-FBP-p-XD-SP (8)

FBP-p-XD-SP (7) (0.4030 g, 0.3949 mmol, 1.0 eq) was suspended in DMF andFmoc-6-Ahx-OH (0.4885 g, 1.3822 mmol, 3.5 eq), DIC (0.3058 mL, 1.9747mmol, 5.0 eq) and HOBT (0.1334 g, 0.9874 mmol, 2.5 eq) were added to it.The reaction was agitated for 6 h and was monitored by Kaiser test. Theresin was washed with DMF (3 times) and DCM (3 times). 20% piperidine inDMF was added to the resin and the reaction was agitated for 30 min. Theresin was washed with DMF (2 times), 5% HOBT and DMF (2 times). Theresin was suspended in a mixture of DMSO and DIPEA (4:1). Fluoresceinisothiocyanate (FITC, 0.6150 g, 1.5796 mmol, 4 eq) was added to thereaction and it was agitated overnight. The resin was washed with DMF (3times) and DCM (3 times) to obtain FITC-hexyl-FBP-p-XD-SP. (M+2H)²⁺/2:Expected—1011.4, Observed—1011.3.

3) Synthesis of FITC-Hexyl-Cyclic-FBP-p-XD (9)

Compound 8 was added to a solution of TFA, triisopropylsilane (TIS),ethanedithiol (EDT), and water (92.5:2.5:2.5:2.5, ˜15 mL/gFITC-hexyl-FBP-p-XD-SP). The reaction was stirred for 1 h at roomtemperature. The mixture was filtered and the solution volume reduced.Diethylether was added to precipitate a solid. The mixture wascentrifuged and supernatant was removed. The solid was washed with etherand dried.

The solid was dissolved in a mixture of DMSO and water (50:50) and thepH was increased to 5. The reaction was stirred for 12 h and monitoredby HPLC. The reaction mixture was injected onto a reverse phase C4preparative column. The mobile phase A was a mixture of 0.1% TFA/95%H₂O/5% ACN and mobile phase B was a mixture of 0.1% TFA/10% H₂O/90% ACN.Starting from 0% B, the fraction of B increased to 29% over 10 minutes,then from 29 to 33% B over 15 minutes and then from 33 to 100% B overthe next 3 min. The column was washed with 100% B for 5 min and the % Bwas ramped to 0% in the next 2 min. The system was re-equilibrated at 0%B over 5 minutes (total time=35 min). The peptide eluted at aconcentration of 31% B from the column. The fractions were collected andlyophilized. Yield=0.020 g. (M+2H)²⁺/2: Expected—1010.4,Observed—1010.3%; purity=99.9%.

4) Synthesis of FITC-Hexyl-Cyclic-FBP-p-XD-DOTA (10)

Compound 9 is dissolved in DMF and DOTA(OtBu)₃-ONHS (2 eq) is added tothe solution. The pH of the solution is increased to 8.8 and thereaction stirred for 3 h and monitored by HPLC. The product is dissolvedin a solution of TFA, phenol, MSA, thioanisole and DCM(90:2.5:2.5:2.5:2.5) and allowed to react for 20 min. The solution isthen concentrated under reduced pressure and cold diethylether added toprecipitate a solid. The solid is filtered and then redissolved inACN/water mixture and purified by by reverse phase chromatography.

5) Synthesis of FITC-Hexyl-Cyclic-FBP-p-XD-DOTA-Gd (11)

Compound 10 is dissolved in water and the pH is changed to 6.5 using 1Nsodium hydroxide. Gadolinium (III) chloride hexahydrate (GdCl₃.6H₂O, 1eq) is added to the solution and the pH is adjusted to 6.5 with 1Nsodium hydroxide. The reaction is stirred for 1 h at room temperatureand monitored by HPLC. Residual gadolinium (III) chloride is neutralizedwith ethylenediaminetetraacetic acid (EDTA, 0.25 eq). The reaction isstirred for 15 min. and the pH is adjusted to 6.5 with 1N sodiumhydroxide. The neutralization is monitored by testing with xylenolorange. The product is isolated from solution and purified by reversephase chromatography.

Example 8 Synthesis of Dual PET-Optical Agent,FITC-Hexyl-Cyclic-FBP-p-XD-DOTA-Cu (12)

To a solution of 200 μCi of ⁶⁴CuCl₂ (80 μL) was added 15 μL of 3.1 mMFITC-hexyl-cyclic-FBP-p-XD-DOTA solution (10). The total volume wasincreased to 200 μL by addition of water and 1 M NaOH to adjust the pHto 6.5. The reaction was stirred at 50° C. for 60 min and monitored byreverse phase HPLC with gamma ray detection. The radiochemical purity byHPLC was >98%.

Example 9 Synthesis of Dual SPECT-Optical Agent,FITC-Hexyl-Cyclic-FBP-p-XD-DOTA-in (13)

The In-111 (InCl₃) is transferred into a vial using a 0.1 M NaOAcsolution, pH 5.5. A 1 mM solution of peptide 10 is prepared in 0.1 MNaOAc, pH 5.5. 50 μL of the peptide 10 solution is added to the In-111solution and stirred at 50° C. for an hour. The solution is checked byRP-TLC. If the reaction is not complete and free In-111 is observed,more peptide is added to the reaction mixture and the reaction mixtureis stirred for another 30 min at 50° C. The solution is checked byRP-TLC and this procedure is repeated until almost no free In-111 isleft. The solution is diluted in saline before injection into animals.

Example 10 Synthesis of Dual MR-Optical Agent,DOTA-Cyclic-FBP-p-XD-FITC-Gd (17)

1) Synthesis of DOTA-FBP-p-XD (14)

Compound 7 (0.3224 g, 0.316 mmol, 1.0 eq) was suspended in DMF andDOTA(OtBu)₃-OH (0.5429 g, 0.9479 mmol, 3 eq),2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HATU, 0.6008 g, 1.58 mmol, 5.0 eq) and DIPEA (0.550mL, 3.16 mmol, 10 eq) were added to it. The reaction was agitated for 5h and reaction checked by the Kaiser test. The resin was washed with DMF(3 times) and DCM (3 times).

The resin was added to a solution of TFA, TIS, EDT and water(92.5:2.5:2.5:2.5). The reaction was stirred for 1 h at roomtemperature. The mixture was filtered and the solution volume reduced.Diethylether was added to precipitate a solid. The mixture wascentrifuged and supernatant was removed. The solid was washed with etherand dried. The solid obtained was dissolved in a solution of TFA,phenol, MSA, thioanisole and DCM (90:2.5:2.5:2.5:2.5). The reaction wasstirred for 20 min. The solution was evaporated to a smaller volume.Diethylether was added to precipitate a solid. The mixture wascentrifuged and supernatant was removed. The solid was washed with etherand dried under reduced pressure. (M+2H)²⁺/2: Expected—953.4,Observed—952.9.

2) Synthesis of DOTA-Cyclic-FBP-p-XD (15)

Compound 14 was dissolved in a mixture of DMSO and water (60:40) and thepH was increased to 5. The reaction was stirred for 12 h and monitoredby HPLC. The reaction mixture was injected onto a reverse phase C4preparative column. The mobile phase A was a mixture of 0.1% TFA/95%H₂O/5% ACN and mobile phase B was a mixture of 0.1% TFA/10% H₂O/90% ACN.Starting from 0% B, the fraction of B increased to 29% over 10 minutes,then from 29 to 33% B over 15 minutes and then from 33 to 100% B overthe next 3 min. The column was washed with 100% B for 5 min and the % Bwas ramped to 0% in the next 2 min. The system was re-equilibrated at 0%B over 5 minutes (total time=35 min). The peptide eluted off the columnat a concentration of 30.4% B. Yield=0.0253 g (M+2H)²⁺/2:Expected—951.9, Observed—952.4. % purity=87%.

3) Synthesis of DOTA-Cyclic-FBP-p-XD-FITC (16)

Compound 15 is dissolved in 0.05 M borate buffer, pH 9. FITC (2 eq)dissolved in DMF is added to the DOTA-cyclic-FBP-p-XD solution. Thereaction is stirred for 3 h and monitored by HPLC. The peptide ispurified by reverse phase chromatography.

4) Synthesis of GdDOTA-Cyclic-FBP-p-XD-FITC (17)

Compound 16 is dissolved in water and the pH is changed to 6.5 using 1Nsodium hydroxide. Gadolinium (III) chloride hexahydrate (GdCl₃.6H₂O, 1eq) is added to the solution and the pH is adjusted to 6.5 with 1Nsodium hydroxide. The reaction is stirred for 1 h at room temperatureand monitored by HPLC. Residual gadolinium (III) chloride is neutralizedwith ethylenediaminetetraacetic acid (EDTA, 0.25 eq). The reaction isstirred for 15 min. and the pH is adjusted to 6.5 with 1N sodiumhydroxide. The neutralization is monitored by testing with xylenolorange. The product is isolated from solution and purified by reversephase chromatography.

Example 11

Fibrinogen (fgn) (50 mg, American Diagnostica) was dissolved in 1 mL ofTBS (50 mM Tris, 150 mM sodium chloride, pH 7.4) and was dialyzedagainst TBS with 5 mM sodium citrate. The concentration of the resultingfibrinogen solution was 8.8 mg/mL based on the absorbance at 280 nmwhere 1 mg/mL fgn has an optical density of 1.512 for a 1 cm pathlength. Solutions were prepared by diluting stock solutions ofgadolinium probe and fgn to final solutions of 25 μM Gd and 5 mg/mL fgn.Gadolinium concentrations were determined by ICP-MS. Fibrin gels wereprepared by adding CaCl₂ and thrombin to a final concentration of 7 mMand 4 U/mL, respectively. After briefly mixing the CaCl₂ and thrombin,the solutions were allowed to gel/clot for 30 minutes. After fibrin(fbn) formation was complete, the clots were pushed to the wall of thetube with a pipet tip. A total of 6×0.5 mL phantoms were prepared in 1mL centrifuge tubes: A) pure water; B) 5 mg/mL fibrinogen; C)[Gd(HP-DO3A)] at 25 μM in 5 mg/mL fgn; D) [Gd(HP-DO3A)] at 25 μM in 5mg/mL fbn gel; E) GdDOTA-cyclic-FBP-p-XD-FITC (compound 17) at 25 μM in5 mg/mL fgn; F) GdDOTA-cyclic-FBP-p-XD-FITC (compound 17) at 25 μM in 5mg/mL fbn gel.

All 6 phantoms were simultaneously imaged at room temperature at 1.5 Twith a clinical MRI. T1-weighted gradient echo images were acquired withrepetition time TR=9.2 ms, echo time TE=1.9 ms, flip angle=25°.

The images (FIG. 1) demonstrate that the dual probe is a betterrelaxation agent than commercial GdHP-DO3A and it can be used tovisualize fibrin with MRI. Tubes A and B have similar signal intensity.For the fibrinogen solutions (B, C, E), the presence of gadoliniumincreases the signal intensity (C and E versus B). The tube withGdDOTA-cyclic-FBP-p-XD-FITC (E) is brighter than the tube with GdHP-DO3A(C) indicating that the former is a better relaxation agent. Tube Dshows a uniform signal intensity indicating that the GdHP-DO3A isdistributed equally between the clotted fibrin and the supernatant. Onthe other hand tube F with GdDOTA-cyclic-FBP-p-XD-FITC shows biphasicenhancement. On the right side of tube F the signal is more intense thanin any of the other tubes while on the left side, the signal intensityis decreased relative to tube E. This indicates thatGdDOTA-cyclic-FBP-p-XD-FITC is binding to the fibrin and the fibrin sideof the tube is enriched with Gd resulting in greater signal intensity.

Example 12 Synthesis of Dual PET-Optical Agent,⁶⁴CuDOTA-Cyclic-FBP-p-XD-FITC (18)

To a solution of 200 μCi of ⁶⁴CuCl₂ (80 μL) was added 35 μL of 0.872 mMDOTA-cyclic-FBP-p-XD-FITC (16). The total volume was increased to 200 μLby addition of water and 1 M NaOH to adjust the pH to 6.5. The reactionwas stirred at 50° C. for 60 min and monitored by reverse phase HPLCwith gamma ray detection. The radiochemical purity by HPLC was >98%.

Example 13 Phantom Study with PET

⁶⁴CuDOTA was prepared by reacting 200 μCi of ⁶⁴CuCl₂ with DOTA ligand(50 nmol) in 200 μL water at pH 6.5. The pH of the solution wasmaintained at 6.5 and the reaction was stirred at 50° C. for 30 min. Thesolution was then passed through a cation-exchange resin. The resin waswashed with water and the total final volume was 600 μL.

Six phantoms were prepared in 4 mL glass culture tubes. Each phantomcontained fibrinogen (fgn) at a final concentration of 2 mg/mL, 2.3 nmolof peptide or DOTA, a total volume of 2 mL, and a total activity of 150nCi. In 3 of the phantoms, Fibrin gels (fbn) were prepared by addingCaCl₂ and thrombin to a final concentration of 7 mM and 4 U/mL,respectively. Once the clots had formed, the mixture was centrifuged andthe clots settled to the bottom. The 6 phantoms were labeled: A)⁶⁴CuDOTA in fgn; B) ⁶⁴CuDOTA in fbn gel; C)FITC-hexyl-cyclic-FBP-p-XD-⁶⁴CuDOTA (compound 12) in fgn; D)FITC-hexyl-cyclic-FBP-p-XD-⁶⁴CuDOTA (compound 12) in fbn gel E)⁶⁴CuDOTA-cyclic-FBP-p-XD-FITC (compound 18) in fgn; F)⁶⁴CuDOTA-cyclic-FBP-p-XD-FITC (compound 18) in fbn gel.

The 6 tubes were placed in a holder with each tube equidistant from theisocenter and PET data was collected for 15 minutes. PET imaging wasperformed on a human PET head scanner (BrainPET) at 21° C. The BrainPETgantry physical ID and OD are 36 cm and 60 cm, respectively. The axialfield of view (FOV) is 19.25 cm and the transaxial FOV is ˜30 cm. Eachof the 32 PET detector modules consists of six 12×12 LSO crystal arraysread out by a 3×3 array of APDs (Hamamatsu 8664-55, Japan). Theindividual crystal size is 2.5×2.5×20 mm³. The emission data recorded inlist-mode format were sorted in the line of response space andcompressed axially in the sinogram space for fast reconstruction. Thenormalization was calculated from a 64-hour scan of a plane-sourcerotated in the FOV. The images were reconstructed with the ordinarypoisson ordered subsets expectation maximization (OP-OSEM) algorithmfrom prompts, variance reduced random coincidences, and normalization.The reconstructed volume consisted of 153 slices with 256×256 pixels(1.25×1.25×1.25 mm3).

The images (FIG. 2) demonstrate that the dual probes can visualizefibrin with PET. For the fibrinogen solutions (A, C, E), the ⁶⁴Cu isdistributed equally throughout the samples giving rise to uniform signalintensity. When the fibrinogen is clotted (tube B), there was nodifference in the PET image between tubes A and B indicating thatuntargeted ⁶⁴CuDOTA cannot be used to detect fibrin. The ⁶⁴CuDOTA isdistributed equally in the clot (bottom of tube B) and in the liquidabove the clot. On the other hand, the fibrin-targeted dual probes bothdemonstrate an ability to detect fibrin. The ⁶⁴CuDOTA experiment alsodemonstrates that clot formation does not physically trap the molecule.Tubes D and F indicate that radioactivity is centered in the bottom ofeach tube, i.e. in the clot, and very little activity remains in theliquid above the clot.

Example 14 Phantom Study with Fluorescence Imaging

A 10 mM solution of fluorescein was prepared in ethanol by adding 0.0113g of fluorescein to 3 mL EtOH. This solution was diluted in water toprepare a 9.99 μM solution. Also, a dilute solution ofGdDOTA-cyclic-FBP-p-XD-FITC was prepared with a final concentration of8.55 μM. Four phantoms were prepared in 1 mL glass tubes. Each phantomcontained fibrinogen at a final concentration of 2 mg/mL, 50 pmol ofGdDOTA-cyclic-FBP-p-XD-FITC or fluorescein, at a total volume of 1 mL.In two of the phantoms, fibrin gels were prepared by adding CaCl₂ andthrombin to a final concentration of 7 mM and 4 U/mL, respectively. Oncethe clots had formed, the mixture was centrifuged and the clots settledto the bottom. The 4 phantoms were labeled: A)GdDOTA-cyclic-FBP-p-XD-FITC in fgn; B) GdDOTA-cyclic-FBP-p-XD-FITC infbn gel; C) fluorescein in fbn gel D) fluorescein in fgn. The phantomswere imaged with an IVIS Spectrum imager.

The images (FIG. 3) demonstrate that the dual probe can visualize fibrinwith fluorescence imaging. For the fibrinogen solutions (A, D), thefluorescence intensity is distributed equally throughout the samplesgiving rise to uniform signal intensity. When the fibrinogen is clotted(tube B), the dual probe localizes in the fibrin clot and thefluorescence intensity is much higher in the clot. In the liquid aroundthe clot the fluorescence intensity is reduced with respect to thefluorescence in Tube A. On the other hand, when fibrinogen is clotted inthe presence of untargeted fluorescein (tube C), the fluorescenceintensity is the same throughout the sample indicating little or nobinding.

Example 15 Synthesis of Dual SPECT-Optical Agent,¹¹¹InDOTA-Cyclic-FBP-p-XD-FITC (19)

The ¹¹¹InCl₃ is transferred into a vial using a 0.1 M NaOAc solution, pH5.5. A 1 mM solution of peptide 16 is prepared in 0.1 M NaOAc, pH 5.5.50 μL of the peptide 16 solution is added to the ¹¹¹InCl₃ solution andstirred at 50° C. for an hour. The solution is checked by RP-TLC. If thereaction is not complete and free In-111 is observed, more peptide isadded to the reaction mixture and the reaction mixture is stirred foranother 30 min at 50° C. The solution is checked by RP-TLC and thisprocedure is repeated until almost no free In-111 is left. The solutionis diluted in saline before injection into animals.

Example 16 Synthesis of Dual MR-PET Agent, Gd₃-EP-2104R-Cu (21) 1)Synthesis of Gd₃-EP-2104R (20)

The fibrin-targeted MR probe EP-2104R was synthesized as describedpreviously (Overoye-Chan K, Koerner S, Looby R J, et al. J Am Chem Soc.2008 130(18):6025-39). EP-2104R (0.04 g, 8.32 μmol, 1 eq) was dissolvedin 50 mM citric acid, pH 3. 0.666 mL of 50 mM DTPA (33.28 μmol, 4 eq)solution in water was added to the EP-2104R solution and pH wasmaintained at 3. The solution was stirred for 48 h and was monitored byHPLC on a C₄ column. The mobile phase A was 40 mM ammonium phosphate and0.2 mM EDTA. The mobile phase B contained 70% MeOH and 30% A. Based onHPLC analysis, after 48 h, about 5% of the total Gd was removed fromEP-2104R. The peptide was injected onto a C₄ reverse-phase column toremove the DTPA. The mobile phase A for this column was 95% H₂O/5% ACNand mobile phase B was 90% ACN/10% H₂O. The column was washed with A forthe first five minutes. EP-2104R and Gd-depleted EP-2104R were eluted byincreasing the percentage of B and were collected together. The ACN fromthe fractions was evaporated and the fractions were lyophilized. Theproduct obtained weighed 0.0292 g.

2) Synthesis of Gd₃-EP-2104R-Cu (21)

To incorporate copper-64, 5 mg of Gd-depleted EP-2104R (20) wasdissolved in 0.1 mL of water, and 2.5 mCi of ⁶⁴CuCl₂ in 0.2 mL water wasadded. The pH of this solution was increased to 6.5 using 5 M NaOH. Thereaction mixture was stirred at 50° C. for an hour. The reaction wasmonitored by reverse phase HPLC with a gamma detector. The reactionmixture was stirred with 4 equivalents of diethylenetriamine (dien) for30 min at room temperature. The excess dien, free Cu²⁺ and Cu(dien)²⁺were removed by cation exchange chromatography using Dowex-50 resin,sodium form. The radiochemical purity of the final solution was >98% asdetermined by HPLC. The specific activity of the dual PET-MR probe was380±120 μCi/μmol. The solution was diluted with saline before injectioninto animals.

Example 17 Fibrin Binding of Cu-64 Labeled Probes

Fibrinogen from human plasma (Calbiochem) was dialyzed against 50 mMTris, pH 7.4, 150 mM sodium chloride, 5 mM sodium citrate (TBS•citrate).The fibrinogen concentration was adjusted to 5 mg/mL and CaCl₂ was added(7 mM). The fibrinogen solution (50 μL) was dispensed into the wells ofa 96-well polystyrene microplate (Immulon-II). A solution (50 μL) ofhuman thrombin (2 U/mL) in TBS was added to each well to clot thefibrinogen. The plates were incubated at 37° C. and evaporated todryness overnight. The plates were sealed with tape and stored at −20°C. until use. The wells with dried fibrin were incubated at 37° C. withknown concentrations of the probes. The concentration of the unboundprobe was determined by well counting. The concentration of the boundprobe=[total]−[unbound]. The percentage bound probe was given by [(boundprobe)/total probe)]×100.

% bound for Cu₂-bis-(DOTAGA(OH)₄)-FBP-mXD (5)=66%.

% bound for Gd₃-EP-2104R-Cu (21)=98.7%.

Example 18 MR-PET Imaging of Thrombus with Gd₃-EP-2104R-⁶⁴Cu AnimalProtocol.

All animal studies were approved by the Subcommittee on Research AnimalCare at Massachusetts General Hospital. The occlusive thrombus model wasdescribed previously (Zhang R L, Chopp M, Zhang Z G, Jiang Q, and EwingJ R., Brain Res. 1997 766(1-2):83-92). Briefly, male Wistar rats(350-400 g, n=8; Charles River Laboratories, Wilmington, Mass.) wereanaesthetized with isoflurane (1-2% in 70% N₂O and 30% O₂) and bodytemperature was kept at 37.5° C. A day old autologous blood clot wasinjected into the right internal carotid artery (ICA) at the level ofthe middle cerebral artery (MCA). The femoral vein was cannulated forintravenous delivery of the contrast agent. Simultaneous PET and MR wereacquired on a clinical 3T MRI-BrainPET scanner using a home-builtreceive surface coil and a CP transmit coil. After baseline MR scanswere acquired, the dual MR-PET probe was injected at a dose of 11.5μmol/kg (46 μmol Gd/kg, total activity=19.6±5.9 MBq). The animals weresacrificed 2 h post-injection. In total, six animals underwent imagingwhile in two additional animals only biodistribution data was collected.

General MR-PET Imaging Protocol.

The list-mode emission data from PET were rebinned in the sinogram spacefor fast reconstruction. The uncorrected PET volume was firstreconstructed and binary segmented based on an empirically determinedthreshold in soft tissue and air. A uniform linear attenuationcoefficient (0.096 cm⁻¹, corresponding to water at 511 keV) was assignedto all soft tissue voxels and the resulting attenuation map (combinedwith the coil attenuation map) was forward projected to derive theattenuation correction factors in sinogram space. A model-based approachwas used to derive the scatter sinogram. The normalization wascalculated from a 64 hr scan of a plane-source rotated in the FOV. Theimages were reconstructed with the ordinary Poisson ordered subsetsexpectation maximization (OP-OSEM) algorithm using 16 subsets and 6iterations. The reconstructed volume consists of 153 slices with 256×256pixels (1.25×1.25×1.25 mm³). The spatial resolution at the center of thefield of view is approximately 2.5 mm.

The emission data were recorded in list-mode format for approximately 90min (9×10 min frames) after MR-PET probe administration. The dataacquired in each individual frame were processed and images werereconstructed after acquisition for immediate analysis. Images were alsoreconstructed from the data acquired 30-90 minutes post injection.Additionally, thirty 3-minute frames were generated and the traceruptake in structures of interest as a function of time was analyzed.

The MR imaging sequence included MP-RAGE and 3D T1-weighted gradientecho scans with and without inflow saturation. The latter were used fortime of flight (TOF) angiography. The field of view scanned for the 3Dsequences was 85×85 mm², with repetition time (TR), echo time (TE), andflip angle of 32 ms, 5.78 ms and 18°, respectively. The number ofexcitations (NEX) was 1 with a matrix size of 448×448×92 giving aresolution of 0.19 mm³ in a scan time of 5 min, or 9:35 for the blackblood sequence which used inferior and superior saturation to the nullthe inflowing arterial blood. For the MP-RAGE, the parameters used wereinversion time TI=900 ms, TR/TE=2300/4.4, NEX=1, FOV=49×49 mm²,matrix=192×192, slice thickness=0.26 mm for resolution=0.26 mm³.

Tissue and Blood Analysis.

The ipsilateral (containing the thrombus) and contralateral ICA and MCA,cerebral hemispheres, blood, urine, intraabdominal organs, rectusfemoris muscle and femur bone were collected from all the animals,weighed and radioactivity measured on a gamma counter. The percent ofthe injected dose per gram of tissue (% ID/g) was calculated by dividingthe counts of ⁶⁴Cu/g of tissue by the total counts of the injected dose,based on an aliquot of the injected dose, with correction forradioactive decay. Tissues were later homogenized in nitric acid andanalyzed for Gd concentration by inductively coupled plasma-massspectrometry (ICP-MS).

Image Analysis.

MR images were analyzed using OsiriX (www.osirix-viewer.com) by drawingregions of interest (ROI) in the thrombus, contralateral artery andadjacent tissue and quantifying signal intensity (SI). Noise wasquantified as the standard deviation (SD) of the signal measured in theair outside the animal. Contrast to noise ratios (CNR) were calculatedfor the difference between tissue A and tissue B using the followingequation (1).

CNR(tissue A/tissue B)=[SI(tissue A)−SI(tissue B)]/SD(air)  (1)

Signal intensity ratios (SIR) between the thrombus (ipsilateral) andcontralateral vessel were calculated using equation (2) for images pre-and 10 min post-probe administration.

SIR=[SI(ipsilateral)/SI(contralateral)]_(post)/[SI(ipsilateral)/SI(contralateral)]_(pre)  (2)

For PET, the thirty datasets reconstructed from the three-minute frameswere analyzed using Amide (Loening A M, Gambhir S S., Mol Imaging 20032(3):131-7). Volumes of interest (VOI) were drawn in the thrombus and inthe same area on the contralateral side. Average uptake was quantifiedin both VOI for the same slice and the ratio of ipsilateral tocontralateral uptake was calculated. Time activity curves were alsocalculated for the thrombus, contralateral artery, liver and kidney.

Results.

Time of flight MR angiography (FIG. 4, right) showed restricted flow tothe right side of the brain in all animals imaged, consistent with anocclusive thrombus. In all six animals imaged the thrombus was clearlyvisible on black-blood MR images post injection of the dual probe withrelatively high contrast compared to adjacent tissue (FIG. 4, left). TheCNR post probe was much higher than the CNR measured on thepre-injection images (CNR_(thrombus:brain)=3.0±2.0 post vs −2.0±2.2 pre,p<0.005).

This demonstrates that the dual probe can detect thrombi using MRI andthat combining an angiographic image with the image after the targetedprobe is injected provides more certainty as to the precise location ofthe enhancing thrombus.

FIG. 5 shows multiplanar MR black blood pre-probe (FIG. 5C), MR blackblood post probe (FIG. 5B) and PET images post probe (FIG. 5A) fromco-registered data sets for the MR field of view. The focal increasedsignal on the PET images (FIG. 5A) corresponds to a region that ishyperintense on T1-weighted MR post probe injection (FIG. 5B) but isabsent on pre-probe MR (FIG. 5C). The PET data were analyzed bycomputing a signal intensity ratio (SIR_(ipsi:contra)) between thehyperintense lesion (ipsilateral) and an identical contralateral volumeof interest. For PET, SIR_(ipsi:contra)=0.85±0.48, p<0.01 for nullhypothesis of SIR_(ipsi:contra)=1. For the MR data SIR was computedanalogously to the PET data for the pre and post probe scans. The ratioSIR_(post)/SIR_(pre)=1.71±0.35, p<0.01 for null hypothesis ofSIR_(post)/SIR_(pre)=1.

This data (FIG. 5) shows that the dual probe enables thrombus detectionwith both modalities. In PET the thrombus was quite conspicuous but thelower spatial resolution of PET and the lack of anatomical landmarks inthe PET image makes it more difficult to ascertain if the hyperintensesignal is coming from an artery, a vein, or outside the vasculature.Combining this image with high resolution MRI confirms the presence ofthe thrombus and localizes it precisely within the internal carotidartery.

Time-activity volume-of-interest curves for the thrombus andcontralateral artery were calculated from the images. For the thrombus(FIG. 6) activity remains approximately constant over 90 min suggestingfibrin binding, while in the contralateral vessel the signal decays withtime as the probe is cleared from the blood. The results show in FIG. 6indicate that thrombus conspicuity measured by thrombus to backgroundtissue activity is greatest at late time points. This is a benefit ofusing a ⁶⁴Cu labeled probe with a relatively long radioactive half-lifewherein delayed imaging is feasible at several hours post injection.

The ex vivo ⁶⁴Cu and Gd tissue analyses were consistent with the imagingresults. The % ID/g of ⁶⁴Cu was the greatest in the kidney, followed bythe liver and the thrombus. The concentration of ⁶⁴Cu in the thrombuswas at least 4-fold higher than in the contralateral vessel, bloodplasma, brain, or muscle (p<0.005). The gadolinium findings generallymirrored the ⁶⁴Cu results. Outside the kidney, the highest Gdconcentration was in the thrombus and here it was over 6-fold highercompared to the contralateral vessel, blood plasma, brain, or muscle(p<0.0001). These results quantitatively show that the dual probetargets thrombus in vivo.

Example 19 MR-PET Imaging of Thrombus with Gd₃-EP-2104R-⁶⁴Cu (21) in aRabbit Model of Occlusive Arterial Thrombus

The occlusive thrombus model was similar to the one described above.Briefly, a male New Zealand white rabbit (2.5 kg) was sedated with amixture of ketamine and xylazine and anaesthetized with isoflurane (1-2%in 70% N₂O and 30% O₂) and body temperature was kept at 37.5° C. A dayold autologous blood clot was injected into the right internal carotidartery (ICA). The femoral vein was cannulated for intravenous deliveryof the imaging agents. Simultaneous PET and MR were acquired on aclinical 3T MRI-BrainPET scanner using a home-built receive surface coiland a CP transmit coil. After baseline MR scans were acquired, thecompound 21 was administered at a dose of 300 mCi. The blood pool MRcontrast agent gadofosveset was injected at a dose of 0.1 mmol Gd/kg toacquire an angiographic image. FIG. 7 shows a hyperintense PET signal inthe right internal carotid (ICA). MR with a blood pool agent showslocalization of the PET signal to the right ICA. The PET intensity isfocused in an area where the MR angiogram shows an occlusion. Thisdemonstrates the benefit of using two different imaging modalities to 1)identify the thrombus using the probe 21 with the PET image and 2)precisely localize the hyperintense PET image within the vascular tree.

Example 20 Binding to Human Fibrin

Human fibrinogen (American Diagnostica) was dialyzed against 50 mM Tris,pH 7.4, 150 mM sodium chloride, 5 mM sodium citrate (TBS•citrate) priorto use. The fibrinogen concentration was adjusted to 5 mg/mL, and CaCl₂was added (7 mM). The fibrinogen solution (50 μL) was dispensed into thewells of a 96-well polystyrene microplate (Immulon-II). A solution (50μL) of human thrombin (2 U/mL) in TBS was added to each well to clot thefibrinogen and to yield a final fibrin concentration close to 2.5 mg/mL.The plates were incubated at 37° C. and evaporated to dryness overnight.

Solutions of compound ranging from 0.1 to 50 μM were added to each ofthe wells of the dried fibrin microtiter plate, and the plate was shakenfor 2 h. After incubation, solution was removed and the concentration ofunbound compound was determined from its radioactivity or by ICP-MS. Theconcentration of the fibrin bound species, [bound], was determined by[bound]=[total]−[unbound]. The binding data were fit to a stoichiometricbinding model (Nair et al, Angew. Chem. Int. Ed. 2008 47: 4918-4921).

Binding data for the first binding event:

Compound 5, Kd=9.0±1.9 μM

Compound 17, Kd=0.8±0.3 μM

Compound 11, Kd=0.9±0.3 μM

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1.-49. (canceled)
 50. An imaging agent, or a pharmaceutically acceptablesalt form thereof, comprising a fibrin binding peptide bound to twoimaging reporters, wherein: a) the fibrin binding peptide is:

and b) each of the two imaging reporters is:

wherein M is ⁶⁸Ga, or a pharmaceutically acceptable salt form thereof.51. A pharmaceutical composition comprising an imaging agent and apharmaceutically acceptable carrier, wherein the imaging agent comprisesa fibrin binding peptide bound to two imaging reporters, wherein: a) thefibrin binding peptide is:

and b) each of the two imaging reporters is:

wherein M is ⁶⁸Ga, or a pharmaceutically acceptable salt form thereof.52. A method of imaging fibrin in a mammal, the method comprising: a)administering to the mammal an imaging agent comprising a fibrin bindingpeptide bound to two imaging reporters, wherein: i) the fibrin bindingpeptide is:

and ii) each of the two imaging reporters is:

wherein M is ⁶⁸Ga, or a pharmaceutically acceptable salt form thereof;b) acquiring an image of the fibrin of the mammal using a nuclearimaging technique; c) acquiring an anatomical image of the mammal usingmagnetic resonance imaging or computed tomography; and d) overlaying theimages of steps b) and c) to localize the image of fibrin within theanatomical image of the mammal.
 53. The method of claim 3, wherein thenuclear imaging technique is selected from single photon emissioncomputed tomography and positron emission tomography.
 54. The method ofclaim 3, wherein the images of steps b) and c) are acquiredsimultaneously.
 55. The method of claim 3, wherein the method furthercomprises administering to the mammal a second imaging agent, whereinthe second imaging agent does not target fibrin.
 56. The method of claim6, wherein the second imaging agent is an MRI imaging agent selectedfrom the group consisting of: gadoteridol, gadopentetate, gadobenate,gadoxetic acid, gadodiamide, gadoversetamide, gadoversetamide, andgadofosveset; or a CT imaging agent selected from the group consistingof iopamidol, iohexol, ioxilan, iopromide, iodixanol, ioxaglate,metrizoate, and diatrizoate.
 57. An imaging agent, or a pharmaceuticallyacceptable salt form thereof, comprising a fibrin binding peptide boundto two imaging reporters, wherein: a) the fibrin binding peptide is:

and b) each of the two imaging reporters is:

wherein M is ¹¹¹In, or a pharmaceutically acceptable salt form thereof.58. A pharmaceutical composition comprising an imaging agent and apharmaceutically acceptable carrier, wherein the imaging agent comprisesa fibrin binding peptide bound to two imaging reporters, wherein: a) thefibrin binding peptide is:

and b) each of the two imaging reporters is:

wherein M is ¹¹¹In, or a pharmaceutically acceptable salt form thereof.59. A method of imaging fibrin in a mammal, the method comprising: a)administering to the mammal an imaging agent comprising a fibrin bindingpeptide bound to two imaging reporters, wherein: i) the fibrin bindingpeptide is:

and ii) each of the two imaging reporters is:

wherein M is ¹¹¹In, or a pharmaceutically acceptable salt form thereof;b) acquiring an image of the fibrin of the mammal using a nuclearimaging technique; c) acquiring an anatomical image of the mammal usingmagnetic resonance imaging or computed tomography; and d) overlaying theimages of steps b) and c) to localize the image of fibrin within theanatomical image of the mammal.
 60. The method of claim 10, wherein thenuclear imaging technique is selected from single photon emissioncomputed tomography and positron emission tomography.
 61. The method ofclaim 10, wherein the images of steps b) and c) are acquiredsimultaneously.
 62. The method of claim 10, wherein the method furthercomprises administering to the mammal a second imaging agent, whereinthe second imaging agent does not target fibrin.
 63. The method of claim13, wherein the second imaging agent is an MRI imaging agent selectedfrom the group consisting of: gadoteridol, gadopentetate, gadobenate,gadoxetic acid, gadodiamide, gadoversetamide, gadoversetamide, andgadofosveset; or a CT imaging agent selected from the group consistingof iopamidol, iohexol, ioxilan, iopromide, iodixanol, ioxaglate,metrizoate, and diatrizoate.